A. Al Naib

Bovine oocyte vitrification using the Cryotop method: effect of cumulus cells and vitrification protocol on survival and subsequent development.

The ability to successfully cryopreserve mammalian oocytes has numerous practical, economical and ethical benefits, which may positively impact animal breeding programs and assisted conception in humans. However, oocyte survival and development following vitrification remains poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) bovine oocytes, (2) to compare empirical and theoretical vitrification protocols, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. In Experiment 1, cumulus-enclosed and partially-denuded GV and MII oocytes were vitrified in 15% EG+15% Me(2)SO+0.5M sucrose in two steps. In Experiment 2, GV oocytes were vitrified either as above or using theoretical modeling based on permeability and osmotic tolerance characteristics in 30% EG+11.4% trehalose in three steps or 40% EG+11.4% trehalose in four steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA) 1% X-1000, 1% Z-1000 or both in three steps. In Experiment 1, the survival, cleavage and blastocyst rate of cumulus-enclosed oocytes was significantly higher than those of partially-denuded oocytes when vitrified at the GV stage (93.8% vs. 81.3%, 65.8% vs. 47.3%, 11.3% vs. 4.0%, respectively, P<0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% vs. 91.8%, 35.2% vs. 36.8%, 5.0% vs. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited significantly higher developmental competence than those vitrified at the MII stage (P<0.05). In Experiment 2, there were no significant differences in the survival, cleavage and blastocyst rate among three protocols (86.0% vs. 92.8% vs. 91.2%, 44.8% vs. 54.4% vs. 45.6%, 5.0% vs. 5.4% vs. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P<0.05) than non-vitrified control oocytes. In Experiment 3, the presence of ice blockers did not alter the cleavage rate or blastocyst development (P>0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes using Cryotop method and conventional IVF procedure. Theoretical analysis of permeability characteristics and tolerance limits could not explain the low developmental competence of vitrified oocytes.

Effects of human chorionic gonadotrophin administration on Day 5 after oestrus on corpus luteum characteristics, circulating progesterone and conceptus elongation in cattle

The aim of the present study was to test the hypothesis that elevated concentrations of progesterone (P4) resulting from the induction of an accessory corpus luteum (CL) by human chorionic gonadotrophin (hCG) administration on day 5 after oestrus would lead to advanced conceptus elongation on day 14 following embryo transfer on day 7. The oestrous cycles of cross-bred beef heifers were synchronised and animals were randomly assigned to receive either of two treatments: (1) intramuscular injection of 3000 IU hCG on day 5 after oestrus (n=14); or (2) intramuscular injection of saline on day 5 after oestrus (n=13). Ovaries were scanned daily by transrectal ultrasonography to assess CL development. Serum concentrations of P4 were determined from daily blood samples collected from the jugular vein. In vitro-produced bovine blastocysts were transferred to synchronised recipients on day 7 after oestrus (n=15 blastocysts per recipient). Heifers were killed on day 14 after oestrus and the uterus was flushed to recover the embryos. Injection of hCG on day 5 induced ovulation of the dominant follicle in all treated heifers and increased the total area of luteal tissue on the ovary, which was associated with a significant increase (P<0.001) in serum concentrations of P4 from day 7 to day 14. Positive associations were detected between circulating P4 with CL area (within-day correlations ranging from r=0.45 to r=0.67) and total area of luteal tissue (within-day correlations ranging from r=0.65 to r=0.86) Administration of hCG did not affect the proportion of day 14 conceptuses recovered. However, compared with the control group, hCG-treated heifers had increased conceptus length (3.91±1.23 vs. 5.57±1.02 mm, respectively; P=0.06), width (1.00±0.06 vs. 1.45±0.05 mm, respectively; P=0.002) and area (5.71±0.97 vs. 8.31±0.83, respectively; P=0.02). Although numerically greater, mean interferon-τ (IFNT) production in vitro did not differ significantly (P=0.54) between embryos recovered from hCG-treated and control heifers. In contrast, there was a strong positive correlation between individual embryo length (r=0.76; P<0.001) and individual embryo area (r=0.72; P<0.001) and IFNT production. In conclusion, administration of hCG on day 5 after oestrus resulted in the formation of an accessory CL and hypertrophy of the original CL, the result of which was an increase in P4 concentrations from day 7 onwards. These elevated P4 concentrations were associated with an increased conceptus area. Furthermore, conceptus size was highly correlated with IFNT secretion in vitro.

In vitro assessment of sperm from bulls of high and low field fertility

The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 °C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 °C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilize oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalize residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95% CI 39.5 to 75.3) compared to the low fertility (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95% CI 60.9 to 89.4) and 55.3 (95% CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95% CI 21.1 to 49.6) and 24.2 % (95% CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilize oocytes in vitro; however, once fertilization occurred subsequent embryo development was not significantly affected by fertility status.

Membrane permeability characteristics of bovine oocytes and development of a step-wise cryoprotectant adding and diluting protocol.

Membrane permeability is very helpful for the optimization of effective cryopreservation protocols. In this study, experiments were performed to determine these characteristics for immature (germinal vesicle (GV)) and in vitro matured (metaphase II (MII)) bovine oocytes within 4-37 degrees C, and a new step-wise adding and diluting protocol for ethylene glycol (EG) was developed and verified. Osmotically inactive volumes (V(b)) of GV and MII oocytes were calculated to be 16.1% and 26.1%. The membrane permeability of the oocytes to water (L(p)) in the presence of EG were between 0.08-0.18 and 0.14-0.28 microm/min/atm, and the membrane permeability of the oocytes to solutes (Ps) were between 0.0011-0.0038 and 0.0029-0.0061 cm/min for GV and MII oocytes, respectively. The activation energies (E(a)) for L(p) and P(s) in the presence of EG were 3.68 and 6.84 kcal/mol for GV oocyte, while 3.62 and 0.83-9.08 kcal/mol for MII oocyte. The data indicated that L(p) and P(s) varied significantly between developmental stages and among temperatures evaluated. Based on these results, different protocols for EG adding and diluting from oocytes were developed and tested. The assessment of cleavage rate and embryonic development in vitro confirmed that the designed 4-step adding 2-step diluting protocol indicated a better outcome. The present study is helpful for better understanding of cryobiological properties and the design of cryopreservation protocols for bovine oocytes.

Regulation of non-classical major histocompatability complex class I mRNA expression in bovine embryos

Regulation of expression of the class I major histocompatability complex (MHC class I) at the maternal fetal interface may play a critical role in embryo survival and the establishment of pregnancy in cattle. However, information concerning immunoregulation of implantation in cattle remains quite limited. Therefore, our current research is concerned with characterizing the expression and regulatory effect of a number of immune factors in the developing bovine embryo. We have analysed the effect of embryo culture in vitro (IVC) in medium supplemented with progesterone (P4): leukemia inhibitory factor (LIF), interferon gamma (IFNG), interleukin (IL)-1B, IL3, IL4, IL10 and granulocyte-colony stimulating factor (G-CSF) on in vitro embryo development and expression of the bovine non-classical MHC class I genes NC2, NC3 and N4 in blastocysts. Cytokine supplementation during IVC did not affect cleavage rate or blastocyst development. However, embryo mRNA expression of NC2, NC3 and NC4 was significantly (p≤0.05) modified in a gene- and cytokine-specific manner. Sequence analysis of the promoter regions of these genes confirmed the presence of appropriate binding sites through which the cytokine signalling could be mediated. In contrast to the lack of effect on in vitro blastocyst development, the non-classical MHC-I expression data suggests a preferential immunomodulatory role of these cytokines during preimplantation embryo development.

Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm.

Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. The aim of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro. In vitro matured bovine oocytes were inseminated with fresh semen stored in a caprogen-based diluent, with or without prior Percoll separation. Irrespective of sire, storage of fresh semen at ambient temperature for up to 7 d post collection had no effect on cleavage rate or blastocyst development after IVF. In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Higher numbers of fresh sperm migrated through artificial mucus on Day 0 (day of semen collection) compared with frozen-thawed sperm. On Day 1 and 2 postcollection there was no difference in the number of sperm migrating through the mucus, but storage of sperm at ambient temperature for longer than 2 d resulted in a significant decline in their ability to penetrate mucus compared with frozen sperm from the same ejaculate. In conclusion, bovine sperm retain the ability to fertilize oocytes in vitro for up to 7 d following storage at ambient temperature. However, the ability of sperm to migrate through artificial mucus in vitro is severely depressed after 2 d storage which may have significant implications for the ability of these sperm to reach the site of fertilization in vivo after AI.